Luria Broth media
1 gm Tryptone
0.5 gm Yeast extract
1 gm Sodium chloride
Deionised water 75 ml
Shake all the solutes until dissolved. Adjust the pH to 7.0 with 5 N NaOH.
Bring the volume of the solution to 100 ml with deionised water and
sterilize it by autoclaving it for 20 minutes at 15 psi.

Procedure

Isolation of Plasmid DNA

Pick up a colony of bacteria and inoculate it in a conical flask
containing 100 ml autoclaved Luria broth media supplemented with
antibiotic (Ampicillin 100 µg/ml) and incubate overnight in a 37°C
shaking water bath at 250 rpm.

Pour the culture in a 2.0 ml centrifuge tube and centrifuge at 5000
rpm for 20 minutes.

Discard the supernatant and wash the pellet in 1.0 ml double
distilled water. Centrifuge the solution at 1000 rpm for 5 minutes.

Discard the supernatant and resuspend the pellet in 250µl cold
alkaline solution I (stored at 4°C). Mix properly so that the pellet
dissolves.

Add 20 mg of lysozyme in the above solution and mix well. Allow it
to incubate on ice for 15 minutes.

Now add 250 µl of alkaline solution III and incubate it on ice for
15 minutes.

Centrifuge the sample at 5000 rpm for 10 minutes and filter the
supernatant into an autoclaved 2.0 ml centrifuge tube.

Fill the tube with the same amount of ice-cold isopropyl alcohol
(stored at-20°C) and incubate in cold (-20°C) for 30 minutes.

Centrifuge the sample at 5000 rpm for 15 minutes. Discard the
supernatant and air dry the pellet.

Resuspend the pellet in 500 µl TE buffer.

Purification of Plasmid DNA using Cesium Chloride

When the pellet is suspended completely, measure the mass of plasmid DNA
by using a balance. For every gram of plasmid DNA solution, add 1.0 gm of
solid cesium chloride. Warm the solution at 30°C to dissolve the CsCl salt.

Centrifuge the sample at 50,000 rpm for 10-15 hours at 20°C in an
ultracentrifuge.

After the first run is complete remove the tube carefully and place it in
the clamp and insert a needle just below the band. Remove the cap on the
tube and pull out the band with the help of needle and syringe.

Place the sample in another ultra centrifuge tube and fill the tube with a
solution of cesium chloride and TE. Spin at 50,000 rpm for 5 hours.

When the run is complete follow the above instructions for removing the
bands.

Place the bands in a centrifuge tube and add equal volumes of TE and mix
well. Now add isoamyl alcohol twice the volume of sample in the tube. Shake
well and allow the phases to separate.

Pipet off the top pink layer and repeat the extraction with isoamyl alcohol
until the top layer is clear and transparent.

Dialysis of Plasmid DNA

Prepare pieces of dialysis tubing by rinsing very well in autoclaved
distilled water. Tie one end of the tube and carefully suspend he extracted
plasmid DNA band into the tubing. Tie the other end too and place the sample
in dialysis buffer.

Dialyze for at least 6 hours at 4°C using a magnetic stirrer. Repeat the
process in new buffer solution.

Once the dialysis is complete, remove the liquid from the tubing and
transfer it into another tube.

Add 1/10 volume of 5 M NaCl and twice the volume of pure ethanol.

Centrifuge the samples for 20 minutes at 5000 rpm at 4°C.

Discard the supernatant, leaving the pellet.

Rinse with 95% Ethanol and resuspend the pellet in 100 μl of TE buffer (pH
8.0) or autoclaved distilled water.